Feline Calicivirus Capsid Protein Research Articles

The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.

Inactivation of virus in solution by cold atmospheric pressure plasma: identification of chemical inactivation pathways

Cold atmospheric pressure plasma (CAP) inactivates bacteria and virus through in situ production of reactive oxygen and nitrogen species (RONS). While the bactericidal and virucidal efficiency of plasmas is well established, there is limited knowledge about the chemistry leading to the pathogen inactivation. This article describes a chemical analysis of the CAP reactive chemistry involved in the inactivation of feline calicivirus. We used a remote radio frequency CAP produced in varying gas mixtures leading to different plasma-induced chemistries. A study of the effects of selected scavengers complemented with positive control measurements of relevant RONS reveal two distinctive pathways based on singlet oxygen and peroxynitrous acid. The first mechanism is favored in the presence of oxygen and the second in the presence of air when a significant pH reduction is induced in the solution by the plasma. Additionally, smaller effects of the H2O2, O3 and produced were also found. Identification of singlet oxygen-mediated 2-imidazolone/2-oxo-His (His +14 Da)—an oxidative modification of His 262 comprising the capsid protein of feline calicivirus links the plasma induced singlet oxygen chemistry to viral inactivation.

Investigation of the antiviral properties of copper iodide nanoparticles against feline calicivirus

This study demonstrated the antiviral properties of copper iodide (CuI) nanoparticles against the non-enveloped virus feline calicivirus (FCV) as a surrogate for human norovirus. The effect of CuI nanoparticles on FCV infectivity to Crandell-Rees feline kidney (CRFK) cells was elucidated. The infectivity of FCV to CRFK cells was greatly reduced by 7 orders of magnitude at 1000μgml(-1) CuI nanoparticles. At the conditions, electron spin resonance (ESR) analysis proved hydroxyl radical production in CuI nanoparticle suspension. Furthermore, amino acid oxidation in the viral capsid protein of FCV was determined by nanoflow liquid chromatography-mass spectrometric (nano LC-MS) analysis. The use of CuI nanoparticles showed extremely high antiviral activity against FCV. The high antiviral property of CuI nanoparticles was attributed to Cu(+), followed by ROS generation and subsequent capsid protein oxidation. CuI nanoparticles could be proposed as useful sources of a continuous supply of Cu(+) ions for efficient virus inactivation. Furthermore, this study brings new insights into toxic actions of copper iodide nanoparticles against viruses.

Structural Insights into Calicivirus Attachment and Uncoating

The Caliciviridae family comprises positive-sense RNA viruses of medical and veterinary significance. In humans, caliciviruses are a major cause of acute gastroenteritis, while in animals respiratory illness, conjunctivitis, stomatitis, and hemorrhagic disease are documented. Investigation of virus-host interactions is limited by a lack of culture systems for many viruses in this family. Feline calicivirus (FCV), a member of the Vesivirus genus, provides a tractable model, since it may be propagated in cell culture. Feline junctional adhesion molecule 1 (fJAM-1) was recently identified as a functional receptor for FCV. We have analyzed the structure of this virus-receptor complex by cryo-electron microscopy and three-dimensional image reconstruction, combined with fitting of homology modeled high-resolution coordinates. We show that domain 1 of fJAM-1 binds to the outer face of the P2 domain of the FCV capsid protein VP1, inducing conformational changes in the viral capsid. This study provides the first structural view of a native calicivirus-protein receptor complex and insights into the mechanisms of virus attachment and uncoating.

Apoptosis in cultured cells infected with feline calicivirus.

Caliciviruses are important pathogens of man and animals; feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. To date, little is known about the mechanism of cell damage induced by these viruses. We set out to determine if apoptosis played any role in cell death in FCV infection of cultured cells. We demonstrate that caspase-2, -3, and -7 were activated during FCV infection, as evidenced by pro-form processing and an increase in acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity, as well as cleavage of poly(ADP-ribose)polymerase. Caspase activation coincided with the condensation of chromatin. At about 8 h post infection we also detected cleavage of the FCV capsid protein; this was prevented by caspase inhibitors. Taken together these results suggest that FCV triggers apoptosis within infected cells and that caspases are involved in the cleavage of the capsid protein.

Caspase-mediated cleavage of the feline calicivirus capsid protein.

Feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the 40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-derived FCV capsid protein, revealed that caspase-2, and to a lesser extent caspase-6, cleaved the capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV infection is discussed.

High genetic diversity of the immunodominant region of the feline calicivirus capsid gene in endemically infected cat colonies.

Feline calicivirus (FCV) is an important pathogen of domestic cats. In this study, we have determined the genetic diversity of FCV within four geographically separate colonies of endemically infected cats by sequencing the immunodominant and variable region E of the capsid gene. Comparison of isolates between colonies and between unrelated published sequences gave nucleotide distance values of 26-35% and 22-40%, respectively and suggested each colony was infected with a distinct virus strain. Comparison of isolates within individual endemically infected colonies showed nucleotide distance variability of 0-16%. This was greater than distances previously reported for epidemiologically related isolates from cases of acute disease (0-5%) and was consistent with the evolution of FCV from a single distinct ancestor sequence in each colony. The pattern of nucleotide substitutions generating the observed intra-colony diversity was associated with strong evidence for positive selection acting on immunodominant regions of the FCV capsid protein. We suggest that endemically infected colonies of cats may be important generators of genetic diversity for FCV and that this may ultimately lead to the generation of new strains.

Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein

Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma–FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma–FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat.

Mapping neutralizing and non-neutralizing epitopes on the capsid protein of feline calicivirus.

Neutralizing epitopes on feline calicivirus (FCV) capsid protein were mapped using chimeric capsid proteins recombinant between two FCV isolates that do not show any cross-neutralization. The three chimeric proteins examined were expressed in murine L929 cells employing an MVA/T7 vaccinia virus expression system and inoculated into major histocompatibility complex haplotype-matched C3/HN mice. Based on the neutralizing antibody titre the neutralizing epitope(s) could be mapped to the 5' hypervariable region of the E region or potentially to the C region. The epitopes of some non-neutralizing antibodies were mapped with the same chimeric proteins to the regions B or D and F of the FCV capsid protein.

The capsid gene of feline calicivirus contains linear B-cell epitopes in both variable and conserved regions.

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.

Feline calicivirus capsid protein expression and self-assembly in cultured feline cells

Feline calicivirus (FCV) capsid protein was expressed in feline cells employing the vaccinia virus MVA/T7 RNA polymerase system. The precursor protein was processed to a mature size protein that assembled to virus like particles (VLPs).

Feline calicivirus capsid protein expression and capsid assembly in cultured feline cells.

The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.

The capsid protein of vesicular exanthema of swine virus serotype A 48: relationship to the capsid protein of other animal caliciviruses

Vesicular exanthema of swine virus (VESV), the prototype calicivirus, is the etiologic agent of the porcine disease vesicular exanthema of swine (VES). VES is characterized by vesicle formation on the extremities, mouth and snout and causes abortions and stillbirths if infection occurs during pregnancy. VESV is considered an exotic agent in the US, following its eradication in 1956. The single capsid protein gene of VESV serotype A 48 was cloned and sequenced. The capsid amino acid sequence was 69% similar to the San Miguel sea lion virus serotype 1 (SMSV 1) and 89% similar to the SMSV serotype 4 (SMSV 4) capsid proteins. The six functional regions (A–F) previously identified in SMSV 1, SMSV 4, feline calicivirus and rabbit hemorrhagic disease virus capsid proteins were present in VESV A 48. Two sets of PCR primers were designed which directed amplification of the 5′ end (A region) and the hypervariable (E region) sequences of the capsid protein precursor gene of these viruses, as well as seven additional SMSV serotypes. Alignment and phylogenetic analysis of the N-terminal sequences demonstrated the close relationship of these viruses. Alignment of the hypervariable region amino acid sequences of the ten viruses confirmed that a great variety of sequence exists in this region; however, a consensus sequence (NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R)) was derived which is also present in the feline calicivirus capsid protein. Comparison of the E region sequences provides further evidence that this area of animal calicivirus capsid protein may contain the major antigenic determinants.

Phenotypic and genotypic variation of feline calicivirus during persistent infection of cats

Amino acid sequence of the capsid protein hypervariable region of nine feline calicivirus (FCV) isolates recovered from cats persistently infected after inoculation with the FCV strain 255 parent virus is reported. Capsid proteins from all the isolates were highly cross reactive by Western blot analysis using polyclonal antisera to FCV. Reverse-transcription PCR was used to obtain sequence information of the FCV capsid protein highly variable E region. Amino acid substitutions occurred between residues 426 and 458 of the FCV capsid protein E region. The sequence data and phylogenetic reconstructions based on the sequence information correlated well with antigenic differences among isolates determined by two-way cross neutralization. These results agree with previous reports using divergent isolates of FCV that correlated amino acid differences with serology. This further supports the hypothesis that the FCV capsid protein E region from residues 426 to 458 contains the serotypic determinants of FCV important to antigenic variation.

Mapping of antigenic sites involved in neutralization on the capsid protein of feline calicivirus.

In order to locate amino acid residues involved in the formation of feline calicivirus (FCV) neutralizing epitopes, we analysed the capsid protein gene of monoclonal antibody neutralization-resistant variants of FCV. Amino acid substitutions in the variants were identified in the two hypervariable regions of the capsid protein. Four linear and two conformational epitopes were located in the regions from residues 426 to 460 and 490 to 520, respectively. The relative positions of individual epitopes agreed with our previous antigenic analysis. Two antigenic sites composed of the neutralizing epitopes were mapped in the hypervariable regions of the capsid protein, demonstrating that a relationship exists between the genetic variability and antigenic differences in the neutralization of FCV.

Vaccine efficacy of recombinant feline herpesvirus type 1 expressing immunogenic proteins of feline calicivirus in cats.

We previously constructed a recombinant feline herpesvirus type 1 (FHV1), C7301dlTK-Cap, which contains an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain in the deleted locus of the thymidine kinase (TK) deficient mutant (C7301dlTK) of FHV1. In this report, we carried out in vivo experiments to assess the vaccine efficacy of the recombinant C7301dlTK-Cap against FCV and FHV1 infections in cats. As a result, two vaccinations with the C7301dlTK-Cap by intraocular, intranasal and oral routes protected cats to a significant degree against subsequent virulent challenges with both parent FCV F4 and FHV1 C7301 strains. The results are applicable for the further development of a new genetically engineered polyvalent vaccine for cats.

Recombinant feline herpesvirus type 1 expressing immunogenic proteins inducible virus neutralizing antibody against feline calicivirus in cats

In this study, an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain was inserted into the deletion locus (SmaI site) of the thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) and the resulting recombinant virus was designated as C7301dlTK-Cap. Expression of the FCV antigens by C7301dlTK-Cap was confirmed by indirect immunofluorescence assay and immunoblot analysis. To assess whether the recombinant virus can induce virus neutralizing (VN) antibody against FCV in the natural host, three cats were inoculated intranasally and orally with C7301dlTK-Cap (two cats) or C7301dlTK (one cat). As a result, sera collected from cats inoculated with the C7301dlTK-Cap possessed VN antibody against FCV. This recombinant virus is expected as a new polyvalent recombinant vaccine against FHV-1 and FCV infections.

Characterization of Two Density Populations of Feline Calicivirus Particles

Feline calicivirus (FCV) F9 strain was propagated in Crandall-Reese feline kidney cells. Two density populations of viral particles were observed after equilibrium centrifugation in an isopyknic CsCl gradient. The buoyant density of the heavy particle (PH) is 1.33 g/ml. The light particle (PL), a previously undescribed form of feline calicivirus, has a buoyant density of 1.22 g/ml. The PH and PL presented a similar morphology by electron microscopy. Western blot showed that both PH and PL contained a major polypeptide of the typical FCV capsid protein with a molecular weight of 62,000. Infectivity assay and RNA isolation demonstrated that PH is the intact infectious virion while PL is FCV empty capsid.

Analysis of capsid protein gene variation among divergent isolates of feline calicivirus

Genomic variability within the capsid protein gene of feline calicivirus (FCV) was evaluated among different isolates using hybridization analysis and enzymatic viral nucleic acid amplification. Total infected cell RNA was first hybridized with cDNA clones generated to the capsid gene of the FCV isolates CFI/68, 255, LLK, NADC and KCD. Field isolates of FCV were categorized by hybridization with capsid gene cDNA from the reference strains. Isolates that did not hybridize were positive by Western blot using a cross-reactive cat polyclonal FCV CFI/68 capsid protein antiserum. Using previously published sequence information, oligonucleotide primers were generated based on conserved sequences surrounding the hypervariable capsid protein gene regions. Analysis of the FCV capsid protein gene hypervariable regions was completed by sequencing products of FCV nucleic acid amplified by reverse transcription and polymerase chain reaction. From these data, amino acid substitutions in the hypervariable regions of the capsid protein were identified for those isolates that did not hybridize with the original cDNA clones. An association between phylogenetic relationships and serum neutralization was established among FCV isolates examined.